Tumor Systems
Significant challenges remain in the ability to translate fundamental discoveries in cancer biology and genetics into anti-cancer drug discovery and personalized cancer therapy. Given the high failure (>90%) rate of cancer drugs, there was a need for development of a suitable 3D culture system instead of the currently used monolayer (2D) cultures for anticancer drug development.
To overcome these limitations, researchers have turned to 3D in vitro tumor model systems that better replicate the structure, physiology, and function of tissues, and recreate the in vivo morphology and arrangement of individual cells, concentration gradients of signaling molecules and therapeutic agents and the composition, structure, and mechanical forces of extracellular matrix (ECM) around cells. A variety of approaches such as hanging drop, hydrogels, scaffolds, and spinner flask have been developed to create tumor spheroids, considered the best-validated 3D model, however most of these approaches fail to fully recapitulate in vivo tumors.
The best available 3D tumor spheroid systems suffer from a number of problems including long cultivation time, formation of unequal-sized spheroids, and difficult mechanical accessibility. For-instance, hydrogels may create artificial cell-cell or cell-matrix interactions rendering screening of tumor-stroma-interaction-targeting drugs more difficult. The hanging drop method is non-scaffold based, cumbersome, requires more time for epithelial-to-mesenchymal transition (EMT) and does not fully represent the complexity of the in vivo tumor microenvironment (TME). Despite progress in these models, the fact that only ˜10% of researchers today use 3D systems underlies the unmet need to develop a more economical, faster, and better in vitro 3D tumor model that more closely mimics the in vivo TME.
The inventors previously developed a novel 3D electrospun polymeric nanofibrous scaffold (PNS) on which cancer cells form tight, irregular aggregates referred to as tumoroids, which exhibit EMT and drug responsiveness, similar to in vivo tumors. (Girard, Y. K. et al. PLoS One 8, (2013). PNS can be used to a) develop single cell-line tumoroids (SCTs) or multi-cell tumoroids (MCTs), b) investigate inducible or smart drug release from nanoparticles, and c) the potential to target stromal cells in the TME, which play key roles in drug resistance. Previous research has led to the development of technically simple yet biologically robust PNS-MCTs and -biopsy derived tumoroids (BdTs), which resemble tumors in vivo, and in cancer, can aid in identification of biomarkers of clinical efficacy. The most notable feature of the PNS-MCTs and -BdTs is their characteristic tumor heterogeneity resulting from co-culture of tumor cells with stromal cells, such as cancer-associated fibroblasts (CAFs) and endothelial cells (ECs), the components of TME that participate in inducing drug resistance. However, a major limitation of the PNS-MCT/BdT system is that the data are acquired from PNS-derived MCTs cultured in static media over 7-10 days, which contrasts in vivo conditions, where drugs are in circulation. Also, often parallel wells are used for different time points and dose response studies the data acquired may not provide adequate data due to errors. The static culture conditions distract the tumoroids mimicking in vivo conditions.
Surface Acoustic Wave (SAW) Sensors
Detection and quantification of cell viability and growth in two-dimensional (2D) and three-dimensional (3D) cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up several replicates for time lapse or dose response studies. Currently, cell growth or proliferation of flat 2D cultures utilize MTT assay, flow cytometry and Ki 67 staining. Similarly, measuring cell growth and proliferation in 3D cultures consist of terminal studies that may include trypsinization and staining with trypan blue and quantification. Longitudinal detection of cancer cell viability and growth in 2D and 3D cell cultures in a non-invasive and touch-free fashion remains a major unmet need in research pertaining to cancer cell biology and anti-cancer drug development. The potential application of biosensors for the detection of cell growth has not been reported and remains to be elucidated.
Generally, biosensors such as surface acoustic wave (SAW) are widely used in cancer biomarker detection and bio-agent detection. Gas sensors, biosensors and chemical sensors are a few of the leading applications for surface acoustic wave (SAW) sensors. [(Shen, C. & Liou, S. 131, 673-679 (2008); Onen, O. et al, 12317-12328 (2012); Onen, O. et al. Sensors (Basel). 12, 7423-37 (2012); Vivancos, J.-L. et al, Sensors Actuators B Chem. 171-172, 469-477 (2012)] Generally, biosensors are widely used in cancer biomarker detection and bio-agent detection. Due to SAWs' advantages of low cost, small size and ease of assembly, SAW-based biosensor technologies have the potential to transform the cancer and bio-agent detection fields. (Onen, O. et al. Sensors (Basel). 12, 7423-37 (2012); Pomowski, et al. 15, 4388-4392 (2015))
SAWs include two particle displacement components. One is along the direction of wave propagation and the second one is normal to the surface, such as Rayleigh waves. Rayleigh waves, which generate compressional waves, are affected and damped by the liquid loading and dissipate the wave energy into the liquid. Therefore Rayleigh surface acoustic waves are less sensitive to mass loading changes. [(Nomura, T. et al. Sensors Actuators B Chem. 76, 69-73 (2001)] Shear horizontal surface acoustic waves (SH-SAWs) with the substrate polarized normal to wave propagation are most commonly used in sensor applications that involve fluidics.
Many different wafer types with special cuts are used for shear horizontal wave excitation, such as ST-cut Quartz and 36° Y-cut LiTaO3. [(Nomura, T. et al. Sensors Actuators B Chem. 76, 69-73 (2001); Deobagkar, D. D, et al. 104, 85-89 (2005); Roederer, et al. 2333-2336 (1983); Kondoh, J. et al. 129, 575-580 (2008); Nomura, T., et al. Sensors Actuators B Chem. 91, 298-302 (2003)] ST-cut Quartz and 36° Y-cut LiTaO3 are very stable substrates for sensor applications. However, the electroacoustic coupling coefficient (K2) of ST-cut Quartz is much smaller than that of 36° Y-cut LiTaO3 (36° Y-cut LiTaO3 is 4.7 and ST-cut Quartz is 0.0016). [(Litao, Y. et al. Sensors Actuators A. Phys. 193, 87-94 (2013)] Because of its high electroacoustic coupling coefficient, the 36° Y-cut LiTaO3 generates more stable signals when the SH-SAWs travel through polydimethylsiloxane (PDMS) which absorbs the majority of the energy generated by the interdigital transducers. [(Shilton, R. J, et al. (2014); Li, F. (ProQuest, UMI Dissertation Publishing (Sep. 4, 2011), 2011] PDMS has been widely used in biomedical devices due to its biocompatibility and ease of manufacture into fluidic channels. An optimization of the PDMS channel sidewall thickness was demonstrated to reduce the damping effect of the PDMS on the wave propagation thereby increasing the sensitivity of the sensor. [(Jo, M. C. & Guldiken, R. Microelectron. Eng. 113, 98-104 (2014)]
Even though 36° Y-cut LiTaO3 has a higher electroacoustic coupling coefficient, it also has a higher temperature coefficient compared to the ST-Quartz. Various guide layers can be deposited on the LiTaO3 to change the phase velocity and temperature coefficient of the system. Zinc Oxide (ZnO) is a relatively common material in sensor and SAW fields. The majority of SAW devices coated with ZnO are used as pH or UV sensors. [Oh, H. et al. Microelectron. Eng. 111, 154-159 (2013); Chivukula, V. et al. Appl. Phys. Lett. 96, 3-6 (2010)] Coating a ZnO layer on a LiTaO3 substrate reduces the temperature coefficient and increases the mass sensitivity, hence addressing the shortcoming of the LiTaO3 substrate as opposed to its alternatives. [Powell, D. A. et al. Sensor Actuat A-Phys 115, 456-461 (2004); Powell, D. a., Kalantar-zadeh, K. et al. 2002 IEEE Ultrason. Symp. 2002. Proceedings. 1, 493-496 (2002); Chang, R.-C. et al. Thin Solid Films 498, 146-151 (2006); Fu, Y. Q. et al. Sensors Actuators B Chem. 143, 606-619 (2010)]
For effective biosensing there is a dire need for the development of non-invasive and touch-free detection of cancer cell viability and growth or proliferation in three-dimensional (3D) cell cultures as it pertains to assessing clinical efficacy of anti-cancer drugs. Currently a single platform integrating non-invasive biosensing with a perfused MCT platform does not exist. Having a single platform combining microfluidics for perfusion-based nanodrug delivery, acoustic biosensing with real-time physiologic readouts and MCTs with an in vivo TME provides a unique opportunity to study in vivo nanodrug transport and increase the understanding and highly impact drug delivery to cancer cells and estimating the clinical efficacy of anticancer drugs. However, in view of the art considered as a whole at the time the present invention was made, it was not obvious to those of ordinary skill in the field of this invention how the shortcomings of the prior art could be overcome.